Evening primrose (Oenothera paradoxa) cake as an unconventional protein source.

An efficient procedure of a protein isolate production from the evening primrose cake was developed. The cake is a by-product of oil extraction from seeds by using the cold pressing method. The evening primrose cake contains 22.7% of protein. Its content in the protein isolate derived from the cake is 74%. Proteins present in evening primrose seeds are rich in Trp (7%) and Met (3%), but Lys-deficient (1.3%) as compared to the FAO protein standard. Apart from the proteins, the protein isolate contains 8.5% (w/w per s.s.) dietary fiber, that negatively affects its digestibility. To enhance the bio-availability of the protein isolate, it was partially hydrolyzed with commercial preparations of trypsin and other proteases (Alcalase and Flavourzyme, Novozymes. Denmark). The most advanced proteolysis (52%) was achieved by 6 h digestion of 2% protein suspension with a mixture of Flavourzyme and Alcalase (350 and 600 U per g of protein, respectively) at 50 degrees C and pH 9.0.

Development of an ELISA for detection of myxoma virus-specific rabbit antibodies: test evaluation for diagnostic applications on vaccinated and wild rabbit sera.

An enzyme-linked immunosorbent assay (ELISA) was developed and compared with 2 reference diagnostic tests (indirect immunofluorescence [IF] and complement fixation) to detect myxoma virus-specific antibodies in sera from 50 rabbits experimentally vaccinated with an attenuated strain of myxoma virus or with a Shope fibroma virus. The ELISA was highly specific (100% specificity) and sensitive (100%, 21 days after homologous vaccination). In a comparison of the ELISA with the IF test in 128 wild rabbits from France, discrepant results were obtained in only 11 (8.6%) animals, which were positive with the ELISA and negative with the IF test. The higher sensitivity and the good specificity of the ELISA was confirmed in a serologic survey of 118 rabbits from 2 Kerguelen (Indian Ocean) islands, where the prevalence of myxomatosis varied considerably. The ELISA is an alternative serologic test for diagnosis, vaccine evaluation, and seroepidemiologic surveys of myxomatosis.

Serp2, an inhibitor of the interleukin-1beta-converting enzyme, is critical in the pathobiology of myxoma virus.

Recently, myxoma virus was shown to encode an additional member of the serpin superfamily. The viral gene, called serp2, was cloned, and the Serp2 protein was shown to specifically bind to interleukin-1beta (IL-1beta)-converting enzyme (ICE), thus inhibiting the cleavage of pro-IL-1beta by the protease (F. Petit, S. Bertagnoli, J. Gelfi, F. Fassy, C. Boucraut-Baralon, and A. Milon, J. Virol. 70:5860-5866, 1996). Here, we address the role of Serp2 in the development of myxomatosis, a lethal infectious disease of the European rabbit. A Serp2 mutant myxoma virus was constructed by disruption of the single-copy serp2 gene and insertion of the Escherichia coli gpt gene serving as the selectable marker. A revertant virus was obtained by replacing the E. coli gpt gene by the intact serp2 open reading frame. The Serp2(-) mutant virus replicated with wild-type kinetics both in rabbit fibroblasts and a rabbit CD4(+) T-cell line (RL5). Moderate reduction of cell surface levels of major histocompatibility complex I was observed after infection with wild-type or Serp2(-) mutant myxoma virus, and both produced white pocks on the chorioallantoic membrane of the chick embryo. After the infection of European rabbits, the Serp2(-) mutant virus proved to be highly attenuated compared to wild-type myxoma virus, as demonstrated by the clinical course of myxomatosis and the survival rates of infected animals. Pathohistological examinations revealed that infection with wild-type myxoma virus resulted in a blockade of the inflammatory response at the vascular level. In contrast, rapid inflammatory reactions occurred upon infection with the Serp2(-) mutant virus. Furthermore, lymphocytes in lymph nodes derived from animals inoculated with Serp2 mutant virus were shown to rapidly undergo apoptosis. We postulate that the virulence of myxoma virus in the European rabbit can be partially attributed to an impairment of host inflammatory processes and to the prevention of apoptosis in lymphocytes. The weakening of host defense is directly linked to serp2 gene function and is likely to involve the inhibition of IL-1beta-converting-enzyme-dependent pathways.

[Reaction of the complement fixation test on microtitration plates: application to the serology of myxomatosis. Comparative study of the results with the reaction of indirect immunofluorescence]. / Réaction de fixation du complément en plaques de microtitration: application à la sérologie de la myxomatose. Etude comparative des résultats avec la réaction d'immunofluorescence indirecte.

The authors describe a complement fixation technique on microtitration plates, using an antigen prepared from myxomas induced in rabbits. Compared with indirect immunofluorescence this technique was less cumbersome, more economical, easier to read and (as a conventional procedure) applicable in all laboratories. Results obtained with 165 serum samples tested by both methods showed good correlation and a specificity at least equal to that of indirect immunofluorescence. Taking into account its lower sensitivity, the positive threshold value for complement fixation under the described experimental conditions was a dilution of 1:4 (H50).

Detection of African swine fever virus by a biotinylated DNA probe: assay on cell cultures and field samples.

African swine fever virus was detected in various samples using a molecular hybridization technique. A fragment located in a constant area of the viral genome was biotin-labelled. This probe, when present at a concentration of 100 ng/ml of the hybridization solution, could detect 10 pg of target DNA immobilized on nitrocellulose with cellular DNA and RNA. The virus was evidenced after being passaged on monkey kidney cells, either 8 h post-inoculation (pi) if the multiplicity of infection (MOI) was at least 1 hemadsorbing unit (HAd) per cell, or 24 h later if the inoculum was diluted up to 10(-3) HAd per cell. When passaged on pig leukocytes with a MOI of 0.1 HAd per cell, the virus was evidenced 12 h pi, or 24 h pi with a MOI of 10(-2) HAd per cell. The probe did not hybridize with another DNA virus passaged on cells, neither did it react with non-infected blood or ham, but did so if African swine fever virus was resuspended with the samples. The spleen from uninfected pig and the lymph nodes from a pig which had died from hog cholera were found to be negative, whereas the spleen from a pig which had died of African swine fever was positive. These samples were also tested with a 32P-labelled probe whose sensitivity was 10-fold higher. A non-radioactive probe could be used both for the sensitive and specific diagnosis of African swine fever and the detection of the virus in an epidemiological survey.